Project Summary/Abstract: During the late stages of the virus life cycle, HIV-1 packages two copies of the viral genome into each assembling HIV-1 virus particle. Although this process is critical for production of infectious virus, where and how packaging occurs in cells remains unclear. Because so little is known, it is generally assumed that packaging is initiated when Gag and HIV-1 genomic RNA (gRNA) randomly find each other anywhere in the cytoplasm. In contrast, our findings argue for a very different model, in which gRNA packaging is initiated within a specific subcellular complex through a highly regulated process. Our preliminary data reveal that prior to and during assembly, non-translating gRNA is largely found in cellular complexes termed RNA granules. Moreover, we find that a subclass of RNA granules containing gRNA and Gag can be isolated using an antibody to the cellular ATPase ABCE1. Thus, ABCE1 is a marker for the RNA granules in which packaging takes place. Additionally, we have identified three categories of ABCE1-containing packaging granules: those involved in early, intermediate, and late stages of packaging. In the parent award, we will use our ability to isolate RNA granules representing different stages in HIV-1 packaging to define molecular events involved in initiation and completion of gRNA encapsidation. In Aim 1, inducing Jurkat cells to synchronously express latent HIV-1 genomes will allow us to follow the kinetics of gRNA trafficking through each of the packaging granules we have identified and into released virus. We will also confirm the physiological relevance of HIV-1 packaging granules using HIV-1 infected primary human T cells. In Aim 2, we will use crosslinking-immunoprecipitation with high throughput sequencing (CLIP-seq) to understand conformational changes that occur during packaging. Using CLIP-seq, we will determine whether HIV-1 packaging granules are the sites in which Gag proteins make those changing gRNA contacts. Additionally, chemical crosslinking will be used to identify host proteins in proximity with assembling Gag that can be analyzed subsequently by CLIP-seq to determine if they make contact with gRNA and may be facilitating genome packaging. In the current Administrative Supplement proposal, we request funds to modernize four key equipment systems that we are using currently to achieve the aims of the parent award. These equipment systems are ten to twenty years old. Funding for the proposed new instruments and upgrades would allow us to generate the data required to complete these aims with greater attention to safety, with faster data acquisition speeds, at higher resolution, with modern multiplexing abilities, and using the latest data analysis and quantitation programs. Thus, funding of this equipment supplement will greatly enhance our progress and increase our ability to generate high impact publications from the parent award.